Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis / 生物工程学报

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower Km value and a higher kcat/Km value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.

Improving the activity of creatinase from Alcaligenes sp. KS-85 through semi-rational design / 生物工程学报

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.

Comparison of approaches for site-directed saturation mutagenesis of anarylsulfatase / 西安交通大学学报(医学版)

Objective: To compare the overall efficiency and cost of the construction and screening of libraries of Pseudomonas aeruginosa arylsulfatase (PAAS) through site-directed saturation mutagenesis with random primers (NNN) and precise primers for PCR. Methods: Site-directed mutagenesis libraries were constructed using the random primers (NNN) of the selected site, an equal-mole mixture of precise primers and a purified precise primer each time for the amplification of the fusion vector of Escherichia coli alkaline phosphatase (ECAP) and PAAS through PCR. The libraries were screened in a high-throughput mode through the assay of activity ratios of PAAS/mutants to ECAP after alkaline lysis of host cells. Three approaches for site-directed saturation mutagenesis were compared for their number of monoclones screened, the number of their expected mutants discovered, overall time consumed, overall cost and other pertinent factors. Results: The site-directed saturation mutagenesis of M72 with random primers for PCR resulted in only 10 among 20 expected mutants after the screening of over 600 monoclones, besides obvious codon bias. In contrast, no obvious codon bias was observed and there were already 18 among 20 expected mutants after the screening of less than 150 monoclones for site-directed saturation mutagenesis of G138 with similar random primers. The site-directed saturation mutagenesis of M72 with an equal-mole mixture of precise primers yielded all of the 19 expected mutants after the screening of less than 190 monoclones; the site-directed saturation mutagenesis of M72 with the purified precise primers for PCR one-by-one gave all of the 19 expected mutants after the screening of just 2 monoclones for every expected mutant. The use of the purified precise primers for PCR one-by-one was thus more favorable for the construction of libraries of site-directed saturation mutagenesis for the minimum number of monoclones screened, the least overall time and cost. In comparison to the use of the purified precise primers for PCR one-by-one, the use of random primers or the equal-mole mixture of precise primers for PCR to generate the libraries tolerated much greater overall cost and longer time. Conclusion: The generation and screening of the site-directed saturation mutagenesis libraries with precise primers for PCR one-by-one were more practical in elucidating the sequence-activity relationship while the use of equal-mole mixture of precise primers for PCR was preferable for the screening of positive mutants during site-directed saturation mutagenesis.

Recent advances in directed evolution / 生物工程学报

Screening is the bottleneck of directed evolution. In order to address this problem, a series of novel semi-rational designed strategies have been developed based on combinatorial active-site saturation test and iterative saturation mutagenesis, including single code saturation mutagenesis, double code saturation mutagenesis and triple code saturation mutagenesis. By creation of "small and smart" high qualified mutant libraries and combinatorial mutagenesis of specific sites, these new strategies have been successfully applied in multiparameter optimization, e.g. stereo/regioselectivity and activity. This review summarized recent advances in directed evolution and its applications in biocatalysis field.

Site-saturation Mutagenesis Technology and Its Application in Protein Engineering / 中国生物工程杂志

Site-saturation mutagenesis is a newly-developed technology in protein engineering.By manipulating the encoding genes,it can rapidly obtain the mutants of desired proteins whose target residues are substituted by 19 other common amino acids.Site-saturation mutagenesis could serve not only as a powerful tool in protein engineering,but also as an important method in exploring the structure-function relationship of proteins.Several techniques were summarized to achieve site-saturation mutagenesis and introduce their application status in the protein engineering.The problem and promising future of its application were also discussed.